Presence of prophenoloxidase in the humoral fluid of amphioxus Branchiostoma belcheri tsingtauense

The presence of phenoloxidase (PO) activity in the humoral fluid of amphioxus Branchiostoma belcheri tsingtauense was electrophoretically and spectrophotometrically studied. The enzyme was present in the humoral fluid predominantly as an inactive proenzyme, prophenoloxidase (proPO). The optimum temperature for activation of the proPO ranged from 30 degrees C to 35 degrees C, and the enzyme exhibited optimum activity at pH between 7.0 and 7.5. ProPO in the humoral fluid was readily activated to active form PO by exogenous elicitors such as trypsin, zymosan and LPS. The activation of the proPO by exogenous elicitors was significantly enhanced in the presence of 10 mM Ca2+, but was susceptible to serine protease inhibitors like soybean trypsin inhibitor and p-nitrophenyl-p'-guanidinobenzoate. PAGE revealed a single band of PO activity in the humoral fluid with an apparent molecular mass of 150 kDa, which was resolved to three bands with molecular masses of 44, 46 and 72 kDa, respectively, after SDS-PAGE. This is the first report on the presence of the enzyme PO in amphioxus humoral fluid.     

Xanthine oxidase from human liver: purification and characterization

Xanthine oxidase [EC 1.2.3.2] was purified 2000-fold from human liver. The last step of the procedure involved affinity chromatography. The resulting preparation showed two closely migrating bands of enzyme activity after gel electrophoresis under nondenaturing conditions. No other proteins were detected on these gels. The average particle mass of the enzyme was 300 kDa as determined by size-exclusion chromatography. This together with results of gel electrophoresis under denaturing conditions suggested that the native enzyme was composed of two subunits of approximately 150 kDa each. The electrophoretic patterns also indicated that a portion of these subunits had undergone partial proteolysis. The substrate specificity of the purified human enzyme was studied using an assay in which phenazine ethosulfate coupled the transfer of electrons from the reduced enzyme to cytochrome c. Hypoxanthine, 2-hydroxypurine, xanthine, 2-aminopurine, and adenine were among the most efficient purine substrates studied. Most purine nucleosides tested were oxidized at detectable rates, but with relatively high Km values. The 2'-deoxyribonucleosides were more efficient substrates than were the corresponding ribonucleosides or arabinonucleosides. In a direct comparison with xanthine oxidase from bovine milk, the human enzyme showed a similar specificity toward purine substrates. However, considerable differences between the bovine and human enzymes were observed with nucleoside substrates. With xanthine as the substrate for the human enzyme, 20% of the total electron flow was univalently transferred to oxygen to produce superoxide radicals.     

Purification and characterization of a putative vitellogenin from the ovary of amphioxus (Branchiostoma belcheri tsingtaunese)

An oocyte-yolk protein was purified by double-step chromatography from amphioxus ovaries. The purified protein appeared to exist as a homodimer of approximately 320 kDa in native polyacrylamide gel electrophoresis (PAGE), and was reduced to a single monomer of approximately 160 kDa in sodium dodecyl sulfate-PAGE (SDS-PAGE). The protein was characterized as a phospholipoglycoprotein by native PAGE and staining of gels for phosphorus with methyl green, for lipids with oil red O and Sudan black B, and for carbohydrates using periodic acid/Schiff reagent. In addition, the amino acid composition of the oocyte-yolk protein was generally similar to that of vitellogenins (Vgs) isolated from different phyla of animals including both vertebrates and invertebrates. The purified phospholipoglycoprotein is thus considered as putative amphioxus Vg.     

Purification and Characterization of Cystathionine (gamma)-Lyase from Lactococcus lactis subsp. cremoris SK11: Possible Role in Flavor Compound Formation during Cheese Maturation

A cystathionine (gamma)-lyase (EC 4.4.1.1) ((gamma)-CTL) was purified to homogeneity from a crude cell extract of Lactococcus lactis subsp. cremoris SK11 by a procedure including anion-exchange chromatography, hydrophobic interaction chromatography, and gel filtration chromatography. The activity of SK11 (gamma)-CTL is pyridoxal-5(prm1)-phosphate dependent, and the enzyme catalyzes the (alpha),(gamma)-elimination reaction of L-cystathionine to produce L-cysteine, (alpha)-ketobutyrate, and ammonia. The native enzyme has a molecular mass of approximately 120 to 200 kDa and apparently consists of at least six identical subunits of 20 kDa. In this respect, the SK11 enzyme clearly differs from other bacterial cystathionine lyases, which are all tetrameric proteins with identical subunits of approximately 40 kDa. In addition, the specific activity of purified SK11 (gamma)-CTL toward L-cystathionine is relatively low compared with those reported for other bacterial cystathionine lyases. The SK11 enzyme shows a broad substrate specificity. In the case of L-methionine, the action of SK11 (gamma)-CTL results in the formation of methanethiol, a volatile sulfur compound known to be required in flavor development in cheddar cheese. The (alpha),(beta)-elimination reaction of L-cysteine is also efficiently catalyzed by the enzyme, resulting in the formation of hydrogen sulfide. Although the conditions are far from optimal, cystathionine (gamma)-lyase is still active under cheddar cheese-ripening conditions, namely, pH 5.0 to 5.4 and 5% (wt/vol) NaCl. The possible role of the enzyme in cheese flavor development is discussed.     

Proteomic approach for caudal trauma-induced acute phase proteins reveals that creatine kinase is a key acute phase protein in amphioxus humoral fluid

Elevated creatine kinase (CK) in the circulation was generally regarded to be a passive release from muscle damage. We utilized proteomic methodologies to characterize amphioxus humoral fluid APPs in response to caudal trauma, and found several spots of CK alterations with up-regulation and pI shift. Its amount and enzyme activity showed a dynamic pattern of APP in humoral fluid accompanied with a reduction in enzyme activity of muscle, whereas there was no significant difference in CK amount of muscle and the other tissues and in CK enzyme activity of the other tissues between different time points of sample collection following caudal trauma. In addition, CK phosphorylation regulation during injury was not achieved by monoclonal antibodies separately against phosphothreonine, phosphotyrosine, and phosphoserine. These results suggested that the CK elevation of humoral fluid might be from muscle, being an active response to caudal trauma rather than a passive release from muscle damage. Therefore, CK ability in response to caudal trauma should be highly concerned.     

Purification and characterisation of a novel enantioselective epoxide hydrolase from Aspergillus niger M200

Purification of a novel enantioselective epoxide hydrolase from Aspergillus niger M200 has been achieved using ammonium sulphate precipitation, ionic exchange, hydrophobic interaction, and size-exclusion chromatography, in conjunction with two additional chromatographic steps employing hydroxylapatite, and Mimetic Green. The enzyme was purified 186-fold with a yield of 15%. The apparent molecular mass of the enzyme was determined to be 77 kDa under native conditions and 40 kDa under denaturing conditions, implying a dimeric structure of the native enzyme. The isoelectric point of the enzyme was estimated to be 4.0 by isoelectric focusing electrophoresis. The enzyme has a broad substrate specificity with highest specificities towards tert-butyl glycidyl ether, para-nitrostyrene oxide, benzyl glycidyl ether, and styrene oxide. Enantiomeric ratios of 30 to more than 100 were determined for the hydrolysis reactions of 4 epoxidic substrates using the purified enzyme at a reaction temperature of 10 degrees C. Product inhibition studies suggest that the enzyme is able to differentiate to a high degree between the (R)-diol and (S)-diol product of the hydrolysis reaction with tert-butyl glycidyl ether as the substrate. The highest activity of the enzyme was at 42 degrees C and a pH of 6.8. Six peptide sequences, which were obtained by cleavage of the purified enzyme with trypsin and mass spectrometry analysis of the tryptic peptides, show high similarity with corresponding sequences originated from the epoxide hydrolase from Aspergillus niger LCP 521.     

Purification and properties of Aspergillus niger beta-glucosidase.

Beta-glucosidase was purified from a crude cellulase preparation from Aspergillus niger by affinity chromatography on a methacrylamide-N-methylene-bis-methacrylamide copolymer bearing cellobiamine. The purified enzyme was a dimer with an isoelectric point of 4.0. The molecular mass of the enzyme was estimated to be 240 kDa by gel-permeation chromatography. The enzyme hydrolyzed specifically beta-glucosidic bonds and catalyzed transglucosylation of the beta-glucosyl group of cellobiose to yield 4-O-beta-gentiobiosylglucose in the presence of organic solvents or under neutral conditions.     

Purification and characterization of hemolymph prophenoloxidase from Ostrinia furnacalis (Lepidoptera: Pyralidae) larvae

Prophenoloxidase (PPO) was isolated from the hemolymph of Ostrinia furnacalis larvae and purified to homogeneity. A 369.85-fold purification and 35.34% recovery of activity were achieved by employing ammonium sulfate precipitation, Blue Sepharose CL-6B chromatography and Phenyl Sepharose CL-4B chromatography. The purified enzyme exhibits a band with a molecular mass of 158 kDa on native PAGE and two spots with a molecular mass of 80 kDa and a pI of 5.70, and a molecular mass of 78 kDa and a pI of 6.50, respectively, on two-dimensional gel electrophoresis. The N-terminal amino acid sequences of two subunits are as follows: PPO1, FGEEPGVQTTELKPLANPPQFRRASQLPRD; PPO2, FGDDAGERIPLQNLSQVPQFRVPSQLPTD. The amino acid composition of purified PPO was similar to that from Galleria mellonella. The enzyme kinetic property of the purified protein showed that the affinity of the enzyme for dopamine was higher than that for l-DOPA and N-acetyldopamine. The phenoloxidase (PO) reaction was strongly inhibited by phenylthiourea, thiourea, dithiothreitol and ethylene diamine tetraacetic acid (EDTA), but poorly inhibited by diethyldithiocarbamate (DTC) and triethylenetetramine hexaacetic acid (THAA), and was not inhibited by o-phenanthroline and ethylene glycol-bis (beta-aminoethylether) N,N,N',N'-tetraacetic acid (EGTA). Both Mg(2+) and Cu(2+) stimulated PO activity when compared with controls. The beta-sheet content of PPO treated with Mg(2+) and Cu(2+) increased significantly (P<0.05). The purified PPO has magnesium level of 5.674+/-2.294 microg/mg and copper level of 1.257+/-0.921 microg/mg as determined with ICP-MS, suggesting that the purified PPO is a metalloprotein.     

Purification and substrate specificity of bovine angiotensin-converting enzyme

Angiotensin-converting enzyme was solubilized from bovine lung with detergent and purified over 2300-fold to physical homogeneity by a combination of ammonium sulfate fractionation, molecular sieve chromatography, and ion exchange chromatography. The purified enzyme had an apparent molecular weight of 126,000 in both the denatured, and reduced, denatured forms as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The purified enzyme had a specific activity of 13.6 units/mg. It was inhibited by EDTA and activated by chloride ion. Chloride functioned as a nonessential activator by raising the Vmax 4.26-fold and lowering the KM 5.99-fold under saturating conditions. Under these conditions, the Vmax was 1.2 mumol/min/unit and the KM was 1.3 mM. Three series of peptides having the general structures, Hip-His-X, Hip-X-Leu, and Hip-X-His-Leu were synthesized and used to examine the binding specificity and substrate specificity of the enzyme for amino acids in the COOH-terminal (P'2), penultimate COOH-terminal (P'1), and antepenultimate COOH terminal (P1) peptide positions. These studies indicated that in terms of binding specificity, the relative importance of these three positions was P'2 > P'1 > P1, while the reverse order P1 > P'1 > P'2 was observed for the relative contribution to substrate specificity. Three peptides, Hip-His-D-Leu, Hip-D-His-Leu, and Hip-D-Phe-His-Leu, were also synthesized and used to examine the stereochemical requirements of the enzyme in terms of both peptide binding and hydrolysis. Hydrolysis was found to require an L amino acid in all three positions. In contrast, all three peptides bound to the enzyme.     

Purification and some properties of a novel microbial lactate oxidase

 Geotrichum candidum was found to produce a lactate oxidase. The enzyme was purified by gel filtration and ion-exchange chromatography. The purified lactate oxidase showed a molecular mass of 50 kDa under denaturing and about 400 kDa under non-denaturing conditions. Transmission electron micro-scopy analysis confirmed an octameric structure. FMN was found to be a cofactor for this enzyme. Polarographic studies confirmed an oxygen uptake by the lactate oxidase. The enzyme showed specificity towards the L isomer of lactate and did not oxidise pyruvate, fumarate, succinate, maleate and ascorbate. It was stable at alkaline pH and also for 15 min at 45°C. The addition of glycerol and dextran 500 000 to the enzyme sample enhanced storage stability. Received: 28 September 1995/Received revision: 10 January 1996/Accepted: 15 January 1996     

Purification and characterization of phenoloxidase from brine shrimp Artemia sinica

Phenoloxidase from Artemia sinica (AsPO) was purified by Superdex 200 gel-filtration and Q Sepharose fast flow ion-exchange chromatography, and its properties were characterized biochemically and enzymatically by using L-dihydroxyphenylalanine (L-DOPA) as the specific substrate. Results showed that AsPO was isolated as a monomeric protein of 125.5 kDa in molecular mass. The optimal pH value and temperature are 7.0 and 50°C, respectively, for its PO activity. The AsPO had an apparent K(m) value of 4.2 mM on L-DOPA, and 10.9 mM on catechol, respectively. Oxidase inhibitor on PO activity showed that the AsPO was extremely sensitive to ascorbic acid, sodium sulfite, and citric acid; and was very sensitive to cysteine, benzoic acid, and 1-phenyl-2-thiourea. Combined with its specific enzyme activity on L-DOPA and catechol, it can be concluded that AsPO is most probably a typical catechol-type O-diphenoloxidase. Its PO activity was also sensitive to metal ions and chelators, and 20 mM DETC-inhibited PO activity was obviously recovered by 15 mM Cu(2+), indicating that AsPO is most probably a copper-containing metalloenzyme. All these data about specific substrate, sensitivity to oxidase inhibitor metal ions and chelators indicate that the AsPO has the properties of a catechol-type copper-containing O-diphenoloxidase that functions as a vital humoral factor in host defense via melaninization as in other Crustaceans.     

Purification and characterization of aspartic proteinase from sunflower seeds

Aspartic proteinases were purified from sunflower seed extracts by affinity chromatography on a pepstatin A-EAH Sepharose column and by Mono Q column chromatography. The final preparation contained three purified fractions. SDS-PAGE showed that one of the fractions consisted of disulfide-bonded subunits (29 and 9 kDa), and the other two fractions contained noncovalently bound subunits (29 and 9 kDa). These purified enzymes showed optimum pH for hemoglobinolytic activity at pH 3.0 and were completely inhibited by pepstatin A like other typical aspartic proteinases. Sunflower enzymes showed more restricted specificity on oxidized insulin B chain and glucagon than other aspartic proteinases. The cDNA coding for an aspartic proteinase was cloned and sequenced. The deduced amino acid sequence showed that the mature enzyme consisted of 440 amino acid residues with a molecular mass of 47,559 Da. The difference between the molecular size of purified enzymes and of the mature enzyme was due to the fact that the purified enzymes were heterodimers formed by the proteolytic processing of the mature enzyme. The derived amino acid sequence of the enzyme showed 30-78% sequence identity with that of other aspartic proteinases.     

An unstable small-colony variant of a noninvasive mutant of Salmonella typhimurium is highly invasive for MDCK cells

Abstract A sorbitol dehydrogenase was purified from the membrane fraction of Gluconobacter suboxydans KCTC 2111 (= ATCC 621) by chromatography on CM-, DEAE-, Mono S and Superose 12 columns. The purified enzyme showed a single activity band upon nondenaturing polyacrylamide gel electrophoresis (PAGE) and three subunits of 75, 50 and 14 kDa upon SDS-PAGE. When purified preparations of the enzyme were reconstituted with pyrroloquinoline quinone (PQQ), the specific enzyme activity was significantly increased (up to 9-fold). The absorption spectrum of purified sorbitol dehydrogenase in the reduced state exhibited three absorption maxima (417, 522 and 552 nm) which is in accordance with the typical absorption spectrum of cytochrome c . The 50 kDa subunit appeared as a red band on unstained SDS-gels suggesting its identity as a cytochrome. Fluorescence spectra of extracts from purified sorbitol dehydrogenase showed an excitation maximum at 370 nm and an emission maximum at 465 nm, which conformed to those of authentic PQQ. The purified enzyme showed a rather broad substrate specificity with significant activity toward D-mannitol (68%) and D-ribitol (70%) as well as D-sorbitol (100%). The PQQ-dependent sorbitol dehydrogenase described in this study is clearly different from the FAD-dependent sorbitol dehydrogenase from G. suboxydans var. α IFO 3254 strain in its cofactor requirement and substrate specificity.     

Partial purification and characterization of trehalase from axenically grown myxamoebae of Dictyostelium discoideum

Lysosomal trehalase from the myxamoebae of Dictyostelium discoideum has been partially purified. The behavior of the enzyme under different chromatographic and electrophoretic conditions reveals its close similarities to other lysosomal enzymes that have been studied earlier. The cellular trehalase, which is electrophoretically homogeneous, appears as two peaks of activity when subjected to hydroxyapatite and gel filtration chromatography. The enzyme has isoelectric points of 4.0 and less than 2.5. Among natural disaccharides tested, the purified trehalase showed absolute specificity for trehalose with an apparent Km of 1.15 mM. However, the enzyme efficiently utilized the synthetic sugar alpha-D-glucosyl fluoride as a substrate. Various methods were employed to estimate the apparent molecular weight, which was found to lie in the range of 30-162 kDa.     

Gut CaVP is an innate immune protein against bacterial challenge in amphioxus Branchiostoma belcheri

The importance of calcium-binding proteins in immune response of vertebrates is determined, but whether they have the role in invertebrates is largely unknown. In the present study, phylogenetic analysis indicated that calcium vector protein (CaVP), a protein unique to amphioxus, shared 68% similarity in amino acid sequence with human and mouse calmodulin (CaM). CaVP cDNA was cloned into a bacterial vector pET-32a, and its His-tagged fusion protein was produced in Eschherichia coli cells (BL21). The recombinant CaVP was purified by Ni-NTA column and SDS-PAGE, and then utilized for antibody preparing. The prepared antibodies could recognize amphioxus CaVP with high specificity. Further analysis by Western blotting showed that CaVP was detected in muscle and humoral fluid of normal animals and appeared in gut of bacterial immunized or challenged amphioxus. Interestingly, gut CaVP was significantly higher in a healthy sub-group than a wounded sub-group post bacterial challenge. This response was detected strongly in immunization and challenge by the same Gram-negative bacterium Vibro parahaemolyticus and weakly in immunization by V.?parahaemolyticus and then challenge by Gram-negative Aeromonas hydrophila, whereas no any feedback was found in immunization by V.?parahaemolyticus and challenge by Gram-positive Staphylococcus aureus. These findings indicate the importance of gut CaVP in response to bacterial challenge.     

High-yield purification of a pp60c-src related protein-tyrosine kinase from human platelets

A protein-tyrosine kinase (PTK, EC 2.7.1.112) from human platelets was purified with high yield. Purification of the enzyme involved sequential chromatography on casein-agarose, tyrosine-agarose, heparin-Sepharose and hydroxylapatite. The procedure resulted in substantially enriched 54/52 kDa polypeptides on SDS-polyacrylamide gel electrophoresis and a yield of about 25% in PTK activity. About 250 micrograms of purified protein could be obtained from 1 g of cell protein. The purification factor varied between 1000 and 1500. Determination of the molecular mass of the purified PTK under nondenaturating conditions by molecular sieve chromatography revealed that the enzyme is a monomer of about 50 kDa. Among various protein substrates tested, casein was most prominently phosphorylated. All substrates were exclusively phosphorylated at tyrosine residues. Autophosphorylation at tyrosine residues of the 54/52 kDa proteins was observed in the presence of Mg2+ or Mn2+. At each purification step, the 54/52 kDa proteins were precipitated by sera from tumor-bearing rabbits immunoprecipitating pp60src, but not by control sera. The amount of the immunoprecipitated purified 54/52 kDa phosphoproteins was directly proportional to the amount of antiserum used. Partial peptide mapping by V8 proteinase showed a 26 kDa tyrosine-phosphorylated fragment for the 54 and the 52 kDa proteins as well as for the pp60c-src molecules of intact platelets. All these data indicated that purified PTK is closely related to pp60c-src of human platelets. Using casein as a substrate for the purified enzyme, the Km for ATP was 4 microM and the Vmax for the reaction was 2.0 nmol/min per mg.     

Cloning, expression, purification, and characterization of AmphiTip30, a member of short-chain dehydrogenases/reductases family from the amphioxus Branchiostoma belcheri tsingtauense

In this paper we describe the cloning, expression and identification study of the TIP30 gene from amphioxus (Branchiostoma belcheri). The amphioxus TIP30 cDNA is comprised of 1499 bp and is translated in one open-reading frame to give a protein of 237 amino acids, with a predicted 23 amino acids signal peptide, a 147 bp 5'-UTR and a 638 bp 3'-UTR. A multiple alignment of TIP30 from amphioxus with other known TIP30 sequences shows the conservation of most amino acid residues involved in the peculiar structural domains found within TIP30's. Phylogenetic analysis places AmphiTIP30 at the base of the phylogenetic tree, suggesting that AmphiTIP30 is the archetype of the vertebrate TIP30 genes. We express the amphioxus TIP30 gene in Escherichia coli. driven by T7 promoter. The recombinant amphioxus TIP30 protein was purified by HisTrap affinity column. Subsequently, the binding constant and enzyme activity was mensurated. Western blot and immunohistochemistry analysis confirmed that amphioxus has a native molecular mass of approximately 26 kDa, and TIP30 was strongly expressed in ovary. Finally, the initial function of TIP30 is discussed.     

Purification and characterization of a Z-pro-prolinal-insensitive Z-Gly-Pro-7-amino-4-methyl coumarin-hydrolyzing peptidase from bovine serum--a new proline-specific peptidase.

The study of a new proline-specific peptidase from bovine serum is presented. The enzyme readily cleaves the prolyl oligopeptidase (PO) substrate Z-Gly-Pro-MCA, liberating the fluorophore MCA, thus allowing quantification of enzyme activity. Unlike PO, however, this peptidase is completely insensitive to the PO-specific inhibitor Z-Pro-prolinal and has been designated Z-Pro-prolinal-insensitive Z-Gly-Pro-MCA-hydrolyzing peptidase (ZIP). The two peptidases were successfully separated from each other by phenyl Sepharose hydrophobic interaction chromatography and the subsequent purification focused on the isolation of ZIP from bovine serum. In addition to phenyl Sepharose, calcium phosphate cellulose and DEAE anion-exchange chromatography were employed in the purification, with an overall enzyme yield of 33% and a purification factor of 4023. SDS-PAGE and size-exclusion chromatography indicated a dimeric structure with a relative molecular mass of 174 kDa. The enzyme was stable over the pH range 2.5-10.0. Optimal activity was detected in the pH range 7.4-8.0. Isoelectric focusing revealed a pI of 5.68. Inhibition by AEBSF suggests the peptidase may be a serine protease and ZIP possibly contains a cysteine residue near the active site. alpha(2)M failed to inhibit activity, suggesting oligopeptidase specificity. HPLC analysis revealed a broad substrate specificity for proline-containing peptides. Kinetic analysis indicated that ZIP had a high affinity for Z-Gly-Pro-MCA with a K(m) of 54 microM deduced. Bovine serum ZIP exhibits biophysical characteristics both similar to and different from those of PO isolated from a number of sources and may serve an important physiological function in the degradation of bioactive oligopeptides.     

Purification, characterization and subcellular localization of pig liver alpha-L-iduronidase

alpha-L-Iduronidase was purified about 100,000-fold from pig liver by employing column chromatography on cellulose phosphate (P11), concanavalin A-Sepharose 4B, heparin-Sepharose 4B, Toyopearl HW-55, Sephadex G-100 and chelating Sepharose 6B charged with cupric ions. The molecular mass of the purified enzyme was estimated to be 70 kDa by Sephadex G-100 column chromatography. The purified enzyme gave a single band on disc polyacrylamide gel electrophoresis without using sodium dodecyl sulfate. However, two separate components of 70 kDa and 62 kDa appeared when it was analyzed by SDS/polyacrylamide gel electrophoresis. These 70-kDa and 62-kDa components were confirmed as alpha-L-iduronidase immunochemically. The isoelectric points of these enzymes were both 9.1 as measured by isoelectric focusing in a polyacrylamide gel containing ampholine and sucrose. The optimal pH and Km values were 3.0-3.5 and 65 microM 4-methylumbelliferyl-alpha-L-iduronide, respectively. The purified enzyme was stable in the pH range 3.5-6.0 under conditions with or without 0.5 M NaCl. However, in the presence of 0.5 M NaCl, it was unstable at pH 3.0. Moreover, it was conversely stabilized at pH 7.0 in the presence of 0.5 M NaCl. Immunohistochemically, the enzyme was found in the Kupffer cells and was abundant on their lysosomal membranes. In liver cells, however, the immunohistochemical reaction was weak.     

Purification and characterization of geranylgeranylglyceryl phosphate synthase from a thermoacidophilic archaeon,Thermoplasma acidophilum

We purified a geranylgeranylglyceryl phosphate (GGGP) synthase from Thermoplasma acidophilum by several steps of chromatography. Based on the proteinase-fragment-mass-pattern analysis of the SDS-PAGE band of the partially purified protein, the DNA sequence encoding the protein was identified from the whole genome sequence database of the species. The gene encoding GGGP synthase in T. acidophilum was cloned after PCR amplification of the gene from the genomic DNA. The recombinant enzyme was expressed in Escherichia coli and purified. A single band with a molecular mass of 27 kDa was obtained by SDS-PAGE analysis. The apparent native molecular mass of the enzyme was about 50 kDa based on gel filtration chromatography, suggesting that the enzyme is active as a homodimer. As the GGGP synthase from Methanobacterium thermoautotrophicum has been reported as a pentamer, the enzymes of the two organisms have different oligomeric structures. Other characteristics, including substrate specificity, are similar for the GGGPs of these organisms.